Lack of efficacy of a partial adenosine A1 receptor agonist in neuropathic pain models in mice

Previous studies suggest that adenosine A₁ receptors (A₁R) modulate the processing of pain. The aim of this study was to characterize the distribution of A₁R in nociceptive tissues and to evaluate whether targeting A₁R with the partial agonist capadenoson may reduce neuropathic pain in mice. The cellular distribution of A₁R in dorsal root ganglia (DRG) and the spinal cord was analyzed using fluorescent in situ hybridization. In behavioral experiments, neuropathic pain was induced by spared nerve injury or intraperitoneal injection of paclitaxel, and tactile hypersensitivities were determined using a dynamic plantar aesthesiometer. Whole-cell patch-clamp recordings were performed to assess electrophysiological properties of dissociated DRG neurons. We found A₁R to be expressed in populations of DRG neurons and dorsal horn neurons involved in the processing of pain. However, administration of capadenoson at established in vivo doses (0.03–1.0 mg/kg) did not alter mechanical hypersensitivity in the spared nerve injury and paclitaxel models of neuropathic pain, whereas the standard analgesic pregabalin significantly inhibited the pain behavior. Moreover, capadenoson failed to affect potassium currents in DRG neurons, in contrast to a full A₁R agonist. Despite expression of A₁R in nociceptive neurons, our data do not support the hypothesis that pharmacological intervention with partial A₁R agonists might be a valuable approach for the treatment of neuropathic pain.     

Rapid degradation of dominant-negative Rab27 proteins <Emphasis Type="Italic">in vivo</Emphasis> precludes their use in transgenic mouse models

Background

Transgenic mice have proven to be a powerful system to study normal and pathological gene functions. Here we describe an attempt to generate a transgenic mouse model for choroideremia (CHM), a slow-onset X-linked retinal degeneration caused by mutations in the Rab Escort Protein-1 (REP1) gene. REP1 is part of the Rab geranylgeranylation machinery, a modification that is essential for Rab function in membrane traffic. The loss of REP1 in CHM patients may trigger retinal degeneration through its effects on Rab proteins. We have previously reported that Rab27a is the Rab most affected in CHM lymphoblasts and hypothesised that the selective dysfunction of Rab27a (and possibly a few other Rab GTPases) plays an essential role in the retinal degenerative process.

Results

To investigate this hypothesis, we generated several lines of dominant-negative, constitutively-active and wild-type Rab27a (and Rab27b) transgenic mice whose expression was driven either by the pigment cell-specific tyrosinase promoter or the ubiquitous β-actin promoter. High levels of mRNA and protein were observed in transgenic lines expressing wild-type or constitutively active Rab27a and Rab27b. However, only modest levels of transgenic protein were expressed. Pulse-chase experiments suggest that the dominant-negative proteins, but not the constitutively-active or wild type proteins, are rapidly degraded. Consistently, no significant phenotype was observed in our transgenic lines. Coat-colour was normal, indicating normal Rab27a activity. Retinal function as determined by fundoscopy, angiography, electroretinography and histology was also normal.

Conclusions

We suggest that the instability of the dominant-negative mutant Rab27 proteins in vivo precludes the use of this approach to generate mouse models of disease caused by Rab27 GTPases.

     

On the length of the longest exact position match in a random sequence

A mixed Poisson approximation and a Poisson approximation for the length of the longest exact match of a random sequence across another sequence are provided, where the match is required to start at position 1 in the first sequence. This problem arises when looking for suitable anchors in whole genome alignments.     

Morphological and Biochemical Characterization of the Membranous Hepatitis C Virus Replication Compartment

Like all other positive-strand RNA viruses, hepatitis C virus (HCV) induces rearrangements of intracellular membranes that are thought to serve as a scaffold for the assembly of the viral replicase machinery. The most prominent membranous structures present in HCV-infected cells are double-membrane vesicles (DMVs). However, their composition and role in the HCV replication cycle are poorly understood. To gain further insights into the biochemcial properties of HCV-induced membrane alterations, we generated a functional replicon containing a hemagglutinin (HA) affinity tag in nonstructural protein 4B (NS4B), the supposed scaffold protein of the viral replication complex. By using HA-specific affinity purification we isolated NS4B-containing membranes from stable replicon cells. Complementing biochemical and electron microscopy analyses of purified membranes revealed predominantly DMVs, which contained viral proteins NS3 and NS5A as well as enzymatically active viral replicase capable of de novo synthesis of HCV RNA. In addition to viral factors, co-opted cellular proteins, such as vesicle-associated membrane protein-associated protein A (VAP-A) and VAP-B, that are crucial for viral RNA replication, as well as cholesterol, a major structural lipid of detergent-resistant membranes, are highly enriched in DMVs. Here we describe the first isolation and biochemical characterization of HCV-induced DMVs. The results obtained underline their central role in the HCV replication cycle and suggest that DMVs are sites of viral RNA replication. The experimental approach described here is a powerful tool to more precisely define the molecular composition of membranous replication factories induced by other positive-strand RNA viruses, such as picorna-, arteri- and coronaviruses.     

Post-dispersal seed predation of three grassland species in a plant diversity experiment

Aims Post-dispersal seed predation is an important ecosystem process because it can influence the seed's fate after the initial dispersal from the mother plant and subsequently transform communities. Even at small scales, post-dispersal seed predation can vary greatly depending on seed identity, granivorous taxa or microhabitat structure. However, little is known about the role of plant species richness and functional group richness in post-dispersal seed predation. The overall aim of this study was to test whether increasing plant species richness or plant functional group richness affects the rate and variability of post-dispersal seed predation. We additionally investigated the influence of vegetation structure and seed species identity on the rate and variability of post-dispersal seed predation and whether the influence of different granivorous taxa changed with increasing plant species richness.Methods We conducted seed removal experiments along a long-term experimental plant diversity gradient, comprising plots with monocultures to 60 species mixtures of common grassland species in Jena, Germany, in August 2011. We studied seeds of Onobrychis viciifolia, Pastinaca sativa and Trifolium pratense in exclusion experiments (seed cafeterias), an experimental setup that allowed access either for arthropods or slugs or for all granivorous taxa. Traditionally, seeds removed from seed cafeterias were classified as consumed but we used traceable fluorescent-coloured seeds to obtain more accurate predation rates by subtracting recovered seeds from overall removed seeds. The effect of multiple vegetation variables on mean and variability of seed predation rates was analysed using generalized mixed-effect models and linear regressions, respectively.Important findings Rates of recovered seeds were low but contributed to significant differences between seed predation rates and removal rates of seeds in some treatments. Seed predation rates were not directly correlated with increasing plant species richness or plant functional group richness but were influenced byseed species identity and granivorous taxa. Vegetation variables such as vegetation height and cover were significantly associated with seed predation rates. Depending on the seed species and/or the granivorous taxa, different vegetation variables correlated with seed predation rates. Our results indicate that effects of plant functional group richness and multiple vegetation variables on the magnitude of post-dispersal seed predation varied with seed identity and seed predator taxa. A direct effect of plant species and plant functional group richness could be shown on the variability of post-dispersal seed predation for some seed species and their respective predators. Thus, the changes in magnitude of post-dispersal seed predation with increasing plant species richness could potentially impact the fitness of some plant species and thereby influence plant community structure.     

Alternative splicing coupled mRNA decay shapes the temperature‐dependent transcriptome

Mammalian body temperature oscillates with the time of the day and is altered in diverse pathological conditions. We recently identified a body temperature‐sensitive thermometer‐like kinase, which alters SR protein phosphorylation and thereby globally controls alternative splicing (AS). AS can generate unproductive variants which are recognized and degraded by diverse mRNA decay pathways—including nonsense‐mediated decay (NMD). Here we show extensive coupling of body temperature‐controlled AS to mRNA decay, leading to global control of temperature‐dependent gene expression (GE). Temperature‐controlled, decay‐inducing splicing events are evolutionarily conserved and pervasively found within RNA‐binding proteins, including most SR proteins. AS‐coupled poison exon inclusion is essential for rhythmic GE of SR proteins and has a global role in establishing temperature‐dependent rhythmic GE profiles, both in mammals under circadian body temperature cycles and in plants in response to ambient temperature changes. Together, these data identify body temperature‐driven AS‐coupled mRNA decay as an evolutionary ancient, core clock‐independent mechanism to generate rhythmic GE.     

Deciphering chemotaxis pathways using cross species comparisons

Background  

Chemotaxis is the process by which motile bacteria sense their chemical environment and move towards more favourable conditions. Escherichia coli utilises a single sensory pathway, but little is known about signalling pathways in species with more complex systems.